Project Summary/Abstract Programmed cell death is a critical component of the host antiviral response to infection, serving to limit viral replication, dissemination and spread. Recent attention has revealed programmed necrosis, or necroptosis, as a highly efficient, and important arm of this host response. Necroptosis is controlled by the receptor interacting protein kinase 3 (RIPK3), which receives activating signals arising from ligation of death receptors or pattern recognition receptors (PRRs) to drive rapid cell destruction and limit virus replication. As obligate intracellular pathogens that establish life-long infections, herpesviruses control the fate of the cells they infect, encoding a variety of mechanisms to inhibit the host cell death machinery and promote successful infection. The viral inhibitor of RIP activation (vIRA), encoded by the M45 gene product of murine cytomegalovirus (MCMV), directly inhibits the activation of RIPK3. In the absence of vIRA function, a PRR known as DNA-dependent activator of interferon regulatory factors (DAI/ZBP1) responds to infection and activates RIPK3 to drive necroptosis. While initially described as a cytosolic DNA sensor, accumulating evidence suggests a number of other diverse viruses, including other herpesviruses, influenza and poxviruses, also activate DAI. However, the mechanisms by which they do so remain unclear. Our preliminary studies have revealed that MCMV-induced necroptosis requires viral gene expression mediated by the major immediate early (IE) transactivator, IE3. These data lead to our overall hypothesis that MCMV IE3-dependent transcripts serve as a ligand recognized by DAI to drive antiviral necroptosis, which is inhibited by vIRA during natural infection. This hypothesis will be tested in two specific aims. First, we propose to combine genetic, biochemical and genomics approaches with a set of unique recombinant viruses to define the role of IE3 in necroptosis and identify the ligand(s) it generates to elicit this pathway. Second, we will interrogate and dissect the mechanism by which DAI recognizes MCMV infection and activates RIPK3. The studies proposed here are intended to fill a critical gap in our knowledge of how viruses like MCMV trigger host antiviral necroptosis, and are anticipated to provide insight into how other viruses with significant human health consequences interact with this pathway.